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Hindawi Publishing CorporationInternational Journal of Evolutionary BiologyVolume 2012, Article ID 457264, 16 pagesdoi:10.1155/2012/457264

Review Article

Comparative Pathogenomics of Bacteria CausingInfectious Diseases in Fish

Ponnerassery S. Sudheesh, Aliya Al-Ghabshi, Nashwa Al-Mazrooei, and Saoud Al-Habsi

Microbiology Laboratory, Fishery Quality Control Center, Ministry of Agriculture and Fisheries Wealth,P.O. Box 427, 100 Muscat, Oman

Correspondence should be addressed to Ponnerassery S. Sudheesh, [email protected]

Received 6 February 2012; Accepted 20 March 2012

Academic Editor: Shinji Kondo

Copyright © 2012 Ponnerassery S. Sudheesh et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Fish living in the wild as well as reared in the aquaculture facilities are susceptible to infectious diseases caused by a phylogeneticallydiverse collection of bacterial pathogens. Control and treatment options using vaccines and drugs are either inadequate, inefficient,or impracticable. The classical approach in studying fish bacterial pathogens has been looking at individual or few virulence factors.Recently, genome sequencing of a number of bacterial fish pathogens has tremendously increased our understanding of the biology,host adaptation, and virulence factors of these important pathogens. This paper attempts to compile the scattered literature ongenome sequence information of fish pathogenic bacteria published and available to date. The genome sequencing has uncoveredseveral complex adaptive evolutionary strategies mediated by horizontal gene transfer, insertion sequence elements, mutations andprophage sequences operating in fish pathogens, and how their genomes evolved from generalist environmental strains to highlyvirulent obligatory pathogens. In addition, the comparative genomics has allowed the identification of unique pathogen-specificgene clusters. The paper focuses on the comparative analysis of the virulogenomes of important fish bacterial pathogens, and thegenes involved in their evolutionary adaptation to different ecological niches. The paper also proposes some new directions onfinding novel vaccine and chemotherapeutic targets in the genomes of bacterial pathogens of fish.

1. Introduction

Genome sequencing has provided us with powerful insightsinto the genetic makeup of the microbial world. Themicrobial genomics today has progressed from the longdrawnout individual genome sequencing projects in the pastto a level of technological advancement, where sequencingand comparing the genomes of several strains of a singlepathogen is accomplished in a very short period of time[1, 2]. We are currently passing through a period of explosivedevelopments in the field and an overwhelming glut in thegenome sequence data of microorganisms. To date, over 1800microbial genomes have been published and the sequenc-ing of more than 5200 microbial genome are in differ-ent stages of completion (http://www.ncbi.nlm.nih.gov/gen-omes/lproks.cgi).

The genomics information has categorically disprovedthe earlier thinking that microbial genomes are static and

has demonstrated that genomic evolutionary processes aremuch more flexible and dynamic than previously thought.This has led to the emergence of new ideas such as “uprootingthe tree of life” and the concept of “horizontal genomics”[3–8]. This new thinking about microbial genome evolu-tion has emerged from the observations of lineage-specificgenome reduction and horizontal gene transfer (HGT),frequently occurring in bacterial genomes. Increasingly,genome sequencing projects have identified an unexpectedlevel of diversity among bacteria, which can often be linkedto recombination and gene transfer between a variety ofprokaryotic organisms.

There is large variation in size and content of bacterialgenomes between different genera and species, and alsoamong strains of the same species. Known genome sizes ofbacteria range from under 0.6 to 10 megabases (Mb). Thesmallest bacterial genomes reported are for the mycoplasmasand related bacteria, with sizes as low as 530 kilobases

2 International Journal of Evolutionary Biology

HGT by phages, plasmids, and pathogenicity

islands

Recombination and

rearrangement

Single-nucleotide

polymorphismsPathogen adaptation

Genome reduction in intracellular

niches

Accumulation of pseudogenes

and IS elements

Emergence of genetically

uniform pathogenic

strains

Gene duplication

Core genome

GEIInt

TnGEI

ICE

IS

Plasmid

Phage

Figure 1: Major factors responsible for the pathogenomic evolution of bacteria (modified from [14, 15]; HGT: horizontal gene transfer,GEIs: genomic islands, ICEs: integrative conjugative elements, Int: integrons, Tn: conjugative transposons, IS: IS elements.

[9]. It has been emphasized that the adaptive capability(“versatility”) of bacteria directly correlates with genome size[10].

Genome sequencing of bacterial pathogens has pro-duced exciting information on evolutionary relationshipsbetween pathogenic and nonpathogenic species and hasdemonstrated how each has developed special adaptationsadvantageous for each of their unique infectious lifestyles.In the longer term, an understanding of their genome andbiology will enable scientists to design means of disruptingtheir infectious lifestyles.

The genomes of bacteria are made up of circular or linearchromosomes, extrachromosomal linear or circular plasmidsas well as different combinations of these molecules. Thefunctionally related genes are clustered together in very closeproximity to each other, and those genes located on the“core” part of the chromosome present a relatively uniformG+C content and a specific codon usage. Closely relatedbacteria generally have very similar genomes [11].

The stability and integrity of the “core” sequences ofthe genome, however, is often interrupted by the presenceof DNA fragments with a G+C content and a codon usagemarkedly different from those of the “core” genome. The“flexible” gene pool or the so-called “mobilome” [12],is created by the acquisition of strain-specific “assort-ments” of genetic information mainly represented by mobilegenetic elements (MGE), such as plasmids, bacteriophages,genomic/pathogenicity islands (GEIs/PAIs), integrons, ISelements (ISEs), and transposons (see Figure 1). The flexiblegenes scattered in the genome provide the microbes with anadditional repertoire of arsenal, for example, resistance toantibiotics, production of toxic compounds as well as othervirulence factors [13].

A fundamental question in biology is to define theminimum number of genes or functions to support cellularlife. The size of bacterial genomes is primarily the result oftwo counteracting processes: the acquisition of new genes bygene duplication or by horizontal gene transfer; the deletionof nonessential genes. Genomic flux created by these gainsand losses of genetic information can substantially alter genecontent. This process drives divergence of bacterial speciesand eventually adaptation to new ecological niches [16].

Bacterial pathogens are a major cause of infectiousdiseases and mortality in wild fish stocks and fish rearedin confined conditions. Disease problems constitute thelargest single cause of economic losses in aquaculture [17].Concurrent with the rapid growth and intensification ofaquaculture, increased use of water bodies, pollution, global-ization, and transboundary movement of aquatic fauna, thelist of new pathogenic bacterial species isolated from fish hasbeen steadily increasing [18]. In addition, the virulence andhost range of existing pathogens has also been increasing,posing considerable challenge to fish health researchers,who are actively looking for more efficient vaccines andtherapeutic drugs to combat bacterial fish diseases. Thecurrent treatment methods are ineffective and have manypractical difficulties.

At the level of host-pathogen interaction, there is con-siderable pressure on pathogens to adapt to the harsh hostenvironment as well as to adapt and evolve along with theever changing external environment. The interplay betweenthe host and the pathogen is a complex one, each driven bythe need to secure the success of the species. Adaptations byone partner to exploit new environments will often stimulatethe other to modify its characteristics to take advantage ofthe change. As a consequence of this cycle of interactioncreated by changing environments, new strains of pathogen

International Journal of Evolutionary Biology 3

Table 1: Currently sequenced genomes of bacterial pathogens of fish.

OrganismsSize

(Mb)CDS∗∗

Unknown/Hypothetical

genes (%)

Pseudogenesprophages,

ISE/GEI

%GC

Chromosomes Plasmids

Vibrio anguillarum 775 serotype O1 4.117 3880 26 92 44.3 2 1

Vibrio anguillarum 96F serotype O1 4.065 3766 26 38 42 2 0

Vibrio anguillarum RV22 serotype O2β 4.022 3949 26 68 43.1 2 0

Vibrio ordalii ATCC 33509 3.415 3281 — 31 43.3 2 0

Vibrio harveyi ATCC BAA-1116∗ 6.054 — — — 45.4 2 1

Vibrio vulnificus YJ016 biotype 1 5.26 5028 34 — 46.1 2 1

Vibrio splendidus strain LGP32 4.974 4498 24.8 — 43.8 2 0

Aliivibrio salmonicida strain LFI1238 4.655 4286 — 1179 38.3 2 4

Flavobacterium psychrophilum JIP02/86 2.862 2432 45.3 94 32.5 1 1

Flavobacterium branchiophilum FL-15 3.56 2867 — 54 32.9 1 —

Flavobacterium columnare ATCC 49512∗ 3.2 2896 — — 32.0 1 —

Edwardsiella tarda EIB202 3.76 3486 28 97 59.7 1 1

Edwardsiella ictaluri 93–146∗ 3.812 3783 — 100 57.4 1 —

Aeromonas hydrophila ATCC 7966 4.744 5195 27.7 7 61.5 1 0

Aeromonas salmonicida A449 4.702 4437 — 258 58.5 1 5

Aeromonas veronii Strain B565 4.551 4057 — — 58.7 1 —

A. caviae Ae398 4.43 — — 6 61.4 1 1

Renibacterium salmoninarum ATCC 33209 3.155 3507 25.3 151 56.3 1 0

Streptococcus parauberis 2.143 2641 21.3 — 35.6 1 0

Lactococcus garvieae UNIUD074 2.172 2101 21.8 224 38.7 1 0

Mycobacterium marinum M 6.636 5424 26 65 62.5 1 1∗

Unpublished.∗∗ Coding sequences.

will evolve. Over time, these strains may emerge as newspecies with characteristic disease symptoms.

The use of antibiotics to control fish diseases has metwith limited success and has the potential danger of anti-biotic resistance development in aquatic bacteria (WorldHealth Organization antimicrobial resistance fact sheet 194,http://www.who.int/inf-fs/en/fact194.html) [19]. As aqua-culture is one of the fastest growing food production indus-tries in the world, demand for sustainable ways of combatingfish diseases is gaining significance. There is tremendousscope for developing novel vaccines and therapeutic drugsagainst bacterial fish pathogens.

Genomic evolution and adaptive strategies of bacte-rial fish pathogens are poorly understood and lags farbehind that of human and terrestrial animal pathogens. Adetailed knowledge of the genome sequences of bacterialfish pathogens and how the genomes of the pathogenicspecies or strains evolved from nonpathogenic ancestors orcounterparts will help us better understand their pathogenic-ity mechanisms and strategies of host adaptations. Thisinformation will help identifying novel vaccine and drugtargets in the genomes of pathogens.

Recently, genome sequencing of a number of bacteriapathogenic to fish and other aquatic organisms have beencompleted. The genome sequence and genome character-istics of important bacterial fish pathogens completed andpublished to date are summarized in Table 1.

The main aim of this paper is to put together andsummarize the scattered genome sequencing information onimportant bacterial fish pathogens available in the literatureto date. We sincerely believe that this paper will provide agenomic perspective on the adaptive evolutionary strategiesof bacterial fish pathogens in different ecological niches andwill help better understand the virulence mechanisms andpathogenesis of infections. It is hoped that this will lead tofinding the most appropriate vaccine and therapeutic drugtargets in the genomes and developing efficient control andtreatment methods for fish diseases.

2. Bacterial Pathogens of Fish

Although pathogenic species representing majority of exist-ing bacterial taxa have been implicated in fish diseases,only a relatively small number of pathogens are responsiblefor important economic losses in cultured fish worldwide.Major bacterial pathogens responsible for infectious diseaseoutbreaks in different species of fish are listed in Table 2.Major groups of bacteria causing infectious diseases in fishand the important genome characteristics of these bacteriaare described in the following sections.

3. Vibrios

Bacteria in the genus Vibrio are mainly pathogenic to marineand brackish water fish. However, they are occasionally


Table 2: Major bacterial pathogens of economically important fish.

Causative agent/species Disease Main host fish

Gram-negatives

Vibrio anguillarum VibriosisSalmonids, turbot, sea bass, striped bass, eel,ayu, cod, and red sea bream

Aliivibrio salmonicida(formerly Vibrio salmonicida)

Vibriosis Atlantic salmon, cod

Vibrio vulnificus Vibriosis Eels, tilapia

Vibrio ordalii Vibriosis Salmonids

Vibrio carchariae(syn.: Vibrio harveyi)

Vibriosis, infectious gastroenteritisShark, abalone, red drum, sea bream, seabass, cobia, and flounder

Moritella viscosa(formerly Vibrio viscosus)

Winter ulcer Atlantic salmon

Photobacterium damselae subsp. piscicida(formerly Pasteurella piscicida)

Photobacteriosis(pasteurellosis)

Sea bream, sea bass, sole, striped bass, andyellowtail

Pasteurella skyensis Pasteurellosis Salmonids and turbot

Tenacibaculum maritimum(formerly Flexibacter maritimus)

FlexibacteriosisTurbot, salmonids, sole, sea bass, giltheadsea bream, red sea bream, and flounder

Flavobacterium psychrophilum Coldwater disease Salmonids, carp, eel, tench, perch, ayu

Flavobacterium branchiophila Bacterial gill diseaseA broad range of cultured cold water andwarm water salmonid and nonsalmonidfishes

Flavobacterium columnare Columnaris diseasecyprinids, salmonids, silurids, eel, andsturgeon

Pseudomonas anguilliseptica Pseudomonadiasis, winter disease Sea bream, eel, turbot, and ayu

Aeromonas salmonicida Furunculosissalmon, trout, goldfish, koi and a variety ofother fish species

Aeromonas hydrophilaAeromonas veronii Biovar SobriaAeromonas sobria Biovar Sobria(Motile aeromonads)

Motile aeromonas septicemia (MAS),hemorrhagic septicemia, ulcer disease orred-sore disease, and epizootic ulcerativesyndrome (EUS)

A wide variety of salmonid andnonsalmonid fish, sturgeon, tilapia, catfish,striped bass, and eel

Edwardsiella ictaluri Enteric septicemia Catfish and tilapia

Edwardsiella tarda EdwardsiellosisSalmon, carps, tilapia, catfish, striped bass,flounder, and yellowtail

Yersinia ruckeri Enteric redmouthSalmonids, eel, minnows, sturgeon, andcrustaceans

Piscirickettsia salmonis Piscirickettsiosis Salmonids

Gram-positives

Lactococcus garvieae(formerly Enterococcus seriolicida)

Streptococcosis or lactococcosis Yellowtail and eel

Streptococcus iniae StreptococcosisYellowtail, flounder, sea bass, andbarramundi

Streptococcus parauberis Streptococcosis Turbot

Streptococcus phocae Streptococcosis Atlantic salmon

Renibacterium salmoninarum Bacterial kidney disease Salmonids

Mycobacterium marinum Mycobacteriosis Sea bass, turbot, and Atlantic salmon

reported in freshwater species as well [20, 21]. The distribu-tion of vibriosis is worldwide and causes great economic lossto the aquaculture industry [22]. Vibriosis, one of the majorbacterial diseases affecting fish, bivalves, and crustaceans,is mainly caused by pathogenic species such as Vibrioanguillarum, V. harveyii (Syn. V. carchariae), V. ordalii, andAliivibrio salmonicida (formerly Vibrio salmonicida) [23, 24].

Other species such as V. vulnificus [25, 26] and Moritellaviscosa (formerly Vibrio viscosus) [27] have been implicated

in fish diseases such as septicemia and winter ulcer, respec-tively; more pathogenic species have been isolated frequentlyand reported in the literature [28].

Genome sequences of four major fish pathogenic vib-rios, V. anguillarum, V. ordalii, Aliivibrio salmonicida, andV.vulnificus have been completed and published [29–31].Generally, they have two chromosomes, one larger and onesmaller. The majority of genes that encode cell functionsand pathogenic factors are located in the large one. The


small chromosome usually contains genes for environmentaladaptation.

Vibrio anguillarum is the most studied aetiological agentof vibriosis [32]. V. anguillarum typically causes a hemor-rhagic septicemia. The O1 and O2 serotypes are the virulentstrains frequently isolated from diseased fish [33, 34]. ManyO1 serotype strains harbor 65 kb pJM1-type plasmids, whichcarry the siderophore anguibactin biosynthesis and transportgenes, a main virulence factor of V. anguillarum, while oneof the O1 serotype strains and other serotypes, such as allof the O2 strains, are plasmidless [28, 35, 36]. The O1serotype strains cause disease in salmonid fish, whereas O2 βstrains are usually isolated from cod and other nonsalmonids[28, 32].

Vibrio ordalii is a very close relative of V. anguillarum [37]and was previously recognized as V. anguillarum biotype 2.Vibriosis caused by these two species are strikingly differentbased on histological evidences [38]. V. anguillarum has aspecial affinity for blood and loose connective tissue, whereasV. ordali is mostly present as aggregates in skeletal and cardiacmuscles. V. ordalii has a lesser affinity for blood and developsbacteremia only at late stages of disease.

Genomic sequences of three different strains of V. anguil-larum (the strain 775 containing plasmid pJM1, serotypeO1 strain 96F, and plasmidless serotype O2 β strain RV22)and V. ordali have recently been published [31]. The pJM1plasmid in the strain 775 contains 65 genes including theanguibactin biosynthesis and transport genes that are uniquefor the strain.

V. anguillarum 775 contains more transposase genes(about 53) than 96F (about 23), RV22 (about 42), and V.ordalii (about 18).

The genome comparison of V. anguillarum serotypeshas revealed some interesting differences in the genomiccomposition, indicating horizontal acquisition of virulencegenes and the evolution of different potential virulencemechanisms among the closely related serotypes [31]. TheV. anguillarum 96F strain has a type III secretion system 2(T3SS2) cluster, which is absent in the 775 strain. The T3SS2genes are highly conserved with other T3SS2 genes reportedin V. parahaemolyticus, V. cholera, and V. mimicus [39–41].In the 775 strain, three transposase genes are present at theT3SS2 chromosomal location, one of which probably origi-nated from the pJM1, indicating that the gene cluster is inac-tivated by a transposition, deletion, or inversion event [31].The 775 strain also contains 10 genomic islands includingintegrase, transposase, and some novel sequences conferringgenomic plasticity to adapt to specific ecological niches.

The strain RV22 genome contains the toxin-antitoxinsystems, and genes encoding the accessory V. choleraeenterotoxin (Ace) and the Zonula occludens toxin (Zot),which is not present in the 775 strain. The yersiniabactin-likesiderophore cluster, which is highly conserved in many Vibriospecies and Photobacterium damselae subspecies piscicida[42], is present in strain RV22 and V. ordalii.

A striking feature of V. ordali genome is its significantreduction in size (3.4 Mb) compared to the V. anguillarumstrains 775 (4.1 Mb), 96F (4.0 Mb), and RV22 (4.0 Mb). V.ordali lacks the ABC transporter genes, the type VI secretion

systems, and the gene for microbial collagenase. The Sypbiofilm formation cluster, which is conserved in many Vibriospecies such as V. fischeri, V. vulnificus, and V. parahaemolyti-cus [43, 44], is present only in V. ordalii. Thus, it is probablethat the transition of V. anguillarum to V. ordalii is mediatedby genome reductive evolution to become an endosymbioticorganism; V. ordali has the smallest genome of all vibrios.

Vibrio vulnificus includes three distinct biotypes. Biotype1 strains cause human disease, while biotype 2 infectsprimarily eels, and biotype 3 infections has been associatedwith persons handling Tilapia, although the source andreservoir of biotype 3 have yet to be identified [45]. Inanother classification the terms clade 1 and clade 2 areused based on the multilocus sequence typing (MLST) [46].Biotype 1 strains are present in both clades, whereas biotype2 strains are present only in clade 1, and biotype 3 strainsappear to be a hybrid between clades 1 and 2. Clade 1 strainsare most often isolated from environmental samples, whileclade 2 strains are mostly associated with human disease andare considered more virulent. Recent comparative genomicanalysis of these biotypes or clades has clearly differentiatedthem based on the possession of an array of clade-specificunique genes including the presence of a virulence-associatedgenomic island XII in the highly virulent strains [30].

Aliivibrio salmonicida (formerly Vibrio salmonicida)causes coldwater vibriosis in marine fish such as farmedAtlantic salmon (Salmo salar), sea-farmed rainbow trout(Oncorhynchus mykiss), and captive Atlantic cod (Gadusmorhua) [47]. The Gram-negative bacterium causes tissuedegradation, hemolysis, and sepsis in vivo. Genome sequenc-ing of Aliivibrio salmonicida has revealed a mosaic structureof the genome caused by large intrachromosomal rearrange-ments, gene acquisition, deletion, and duplication of DNAwithin the chromosomes and between the chromosomes andthe plasmids [29].

The genome has many genes that appear to be recentlyacquired by HGT, and large sections of over 300 codingsequences (CDS) are disrupted by IS elements or containpoint mutations causing frame shifts or premature stopcodons [29]. The genomic islands (GIs) identified in thebacteria include major virulence-related genes encodingT6SS and Flp-type pilus and genes that appear to providenew functions to the bacteria. The Tad system has beenproposed to represent a new subtype of T2SS and is essentialfor biofilm formation, colonization, and pathogenesis [48].

The genome analysis has unequivocally confirmed thatAliivibrio salmonicida has undergone extensive rearrange-ment of its genome by losing massive functional genes andacquiring new genes and become host-restricted, allowingthe pathogen to adapt to new niches. IS expansion has beenrelated to genome reduction in the evolution and emergenceof pathogenicity [49], and accumulation of pseudogenes hasbeen described for several other host restricted pathogens[50, 51].

4. Aeromonads

Aeromonas hydrophila and other motile aeromonads areamong the most common bacteria in a variety of aquatic


environments worldwide, including bottled water, chlori-nated water, well water, sewage, and heavily polluted waters,and are frequently associated with severe disease amongcultured and feral fishes, amphibians, reptiles, and birds[52]. Aeromonads are also considered serious emergingpathogens of human beings [53]. Determination of theetiology of diseases involving aeromonad infections hasbeen complicated by the genetic, biochemical, and antigenicheterogeneity of members of this group.

The genus Aeromonas has been conveniently divided intoa group of nonmotile, psychrophilic species, prominentlyrepresented by Aeromonas salmonicida, which is an obli-gate fish pathogen and a second group of mostly humanpathogenic, motile, and mesophilic species including A.hydrophila.

Genome sequencing of A. hydrophila ATCC 7966T ,A. salmonicida subsp. salmonicida A449, A. veronii strainB565, and A. caviae [54–57] has helped in resolving theirtaxonomic confusion and has brought new insights into theway these bacteria adapt to a myriad of ecological niches,their host adaptive evolution and virulence mechanisms.

Aeromonas salmonicida, the causative agent of furuncu-losis in salmonid and nonsalmonid fish, is a non-motile,Gram-negative bacterium; furunculosis is an important dis-ease in wild and cultured stocks of fish inflicting heavy lossesto aquaculture industry worldwide [58, 59]. A. hydrophilacauses a septicemic disease in fish known variously as “motileaeromonas septicemia” (MAS), “hemorrhagic septicemia,”“ulcer disease,” or “red-sore disease” [60]. The disease causedby this bacterium primarily affects freshwater fish such ascatfish, several species of bass, and many species of tropical orornamental fish. A. veronii is the causative agent of bacterialhemorrhagic septicemia in fish and is becoming a majoreconomic problem in the fish-farming industry [23].

Genome sequencing of the fish pathogen A. salmonicidaA449 has confirmed the presence of fully functional genesfor a type III secretion system (T3SS) that has been shown tobe required for virulence in A. salmonicida [61], and genesfor a type VI secretion system (T6SS), which is disruptedby an IS element [55]. The ancestral state of the T3SS inA. salmonicida A449 is ambiguous because of the absenceof the genes in A. hydrophila ATCC 7966T , while other A.hydrophila strains carry T3SS operons on the chromosome[62]. The genome contains a multitude of virulence-relatedgenes including several types of adhesins (e.g., surface layer,flagella, and pili), toxin genes (aerolysin, hemolysin, repeatsin toxin (RTX) protein, and cytolytic delta-endotoxin),secreted enzymes (protease, phospholipase, nuclease, amy-lase, pullulanase, and chitinase), antibiotic resistance genes(tetA, β-lactamase gene, and efflux pumps), and genesinvolved in iron acquisition and quorum sensing.

Most of the above genes are present in A. hydrophilaATCC 7966T genome and an expansion of gene fami-lies (paralogs) of ABC transporters, two-component signaltransduction systems (TCSs), transcriptional regulators, FeScluster-binding proteins involved in energy transduction atthe membrane, and methyl-accepting chemotaxis proteins(MCPs). Interestingly, transposase, resolvase, or insertion

sequence element sequences were not discovered in the A.hydrophila ATCC 7966T genome, whereas these have beenidentified in A. salmonicida and A. caviae genomes. A.salmonicida possesses 88 copies of 10 different IS elementswhereas A. caviae Ae398 has only five different IS elements,and A. hydrophila completely lacks IS elements.

Although A. hydrophila ATCC 7966T has been demon-strated to be the second most virulent species amongAeromonas [63], a very important virulence determinant,T3SS, which is present in A. salmonicida A449 is strikinglyabsent in A. hydrophila ATCC 7966T genome. A. caviaecontains many putative virulence genes, including thoseencoding a type 2 secretion system, an RTX toxin, and polarflagella.

The genome of A. veronii strain B565 contains someputative virulence factors, such as chitinase, RTX protein,adhesion factor, flagella, and mannose-sensitive hemagglu-tinin (MSHA), all of which are shared with A. hydrophilaATCC 7966T and A. salmonicida A449. On the other hand,346 genes including some important putative virulencefactors such as hemolysins and the type III secretion protein,which are shared by the latter two species are absent in A.veronii strain B565.

Many unique genes in A. hydrophila ATCC 7966T andA. salmonicida A449 are virulence genes and often formlarge clusters, such as the rtx cluster in ATCC 7966T andthe flagellar gene cluster in A449, or are involved in mobileelements such as phages and transposons, highlighting theirlateral transfer history [56].

The A. hydrophila ATCC 7966T and A. salmonicidaA449 genomes appear to be very closely related, encodingsimilar number of proteins with only 9% difference in genecontent. However, there are many transposons, phage-relatedgenes, and unique CDS in A. salmonicida A449 genome thatare different from A. hydrophila ATCC 7966T sequences,showing their distinct lineages and adaptive evolution thatoccurred while segregating into different species of the genus.

In sharp contrast to A. hydrophila ATCC 7966T genome,the A. salmonicida A449 genome is characterized by thepresence of large numbers of several different types ofIS elements in multiple copies, with more than 20 genesbeing interrupted by IS elements. A. hydrophila ATCC 7966T

genome has no IS elements.There is a higher tendency for genomic reduction in A.

salmonicida A449 with the formation of many pseudogenes,and A. hydrophila ATCC 7966T has only seven pseudogenes.The formation of pseudogenes has resulted in the lossof function of many genes including flagella and type IVpili, transcriptional regulators, genes encoding carbohydratesynthesis, and modification enzymes and genes for basicmetabolic pathways, which are some characteristic featuresof pathogenomic evolution.

Thus, A. salmonicida A449 appears to have evolvedmuch faster than A. hydrophila ATCC 7966T through geneticrearrangements, genomic reduction, and HGT from com-mon ancestral lineages by acquiring and forming multipleplasmids, prophages, a battery of IS elements, pseudogenes,and several individual genes and operons.


5. Flavobacterium

The genus Flavobacterium includes over 30 species ofwhich Flavobacterium psychrophilum, F. branchiophilum, andF. columnare are important disease agents for salmonids,catfish, and other cultured species [64, 65]. Flavobacteria aresignificant as they are ubiquitous in the soil, freshwater, andmarine environments and are noted for their novel glidingmotility and ability to degrade polymeric organic mattersuch as hydrocarbons [66].

F. psychrophilum is the etiological agent of bacterial cold-water disease (BCWD). It is a serious fish pathogen causingsubstantial economic losses and rearing difficulties to bothcommercial and conservation aquaculture. F. psychrophiluminfections are found throughout the world. Juvenile rainbowtrout and coho salmon are particularly susceptible to BCWD.However, F. psychrophilum infections have been reportedin a wide range of hosts, Anguilla japonica, A. anguilla,Cyprinus carpio, Carassius carassius, Tinca tinca, Plecoglossusaltivelis, Perca fluviatilis, and Rutilus rutilus [64, 67]. Fry andfingerlings with BCWD often have skin ulcerations on thepeduncle, anterior to the dorsal fin, at the anus, or on thelower jaw and mortalities can go up to 70% [68].

F. branchiophilum is the causative organism of bacterialgill disease (BGD) in several parts of the world [69].This disease is characterized by explosive morbidity andmortality rates attributable to massive bacterial colonizationof gill lamellar surfaces and progressive branchial pathologystemming from high rates of lamellar epithelial necrosis [70].

F. columnare (formerly Cytophaga columnaris; Flexibactercolumnaris) is the causative agent of columnaris diseaseof salmonids and other fishes in commercial aquaculture,the ornamental fish industry, and wild fish populationsworldwide [71]. Classically, during outbreaks, its morbidityand mortality rates escalate more gradually than for BGD.Additionally, unlike the pattern of necrosis in BGD, fish withcolumnaris will have severe necrosis of all parts of the gill asthe bacterium invades inwardly [72].

The taxonomy of the three species was initially basedon phenotypic characteristics and has been revised severaltimes during the years. The latest classification based on G+Ccontent, DNA-ribosomal ribonucleic acid (rRNA) hybridi-sation, and fatty acid and protein profiles, has confirmedthat all the three species now belong to the phylum/divisionCytophaga-Flavobacterium-Bacteroides, family Flavobacteri-aceae, and genus Flavobacterium [73].

The whole genome sequences of F. psychrophilum andF. branchiophilum have been published [74, 75]. The F.columnare genome sequence is yet to be completed andpublished [76].

Prominent features of F. psychrophilum infection includethe strong adhesion to fish epithelial tissues followed bygliding motility, rapid and mass tissue destruction, andsevere muscle tissue ulcerations. Hence, the identification ofmultiple genes encoding secreted proteases, adhesins, andgliding motility (gld) genes in F. psychrophilum genomeindicates their possible involvement in the virulence of thepathogen. However, the gene sequence of a secreted colla-genase was disrupted by an insertion sequence of the IS256

family in several strains isolated from rainbow trout [74]indicating the clonal dissemination of strains containingthe disrupted gene. The F. psychrophilum seems to havehorizontally acquired virulence associated genes from otherunrelated bacteria. It has a hemolysin similar to the toxinVAH5, which is a virulence factor in Vibrio anguillarum[77]. It also has a gene encoding a protein that is similarto domains 1–3 of thiol-activated cytolysin family of pore-forming toxins (TACYs), which has been implicated in thepathogenicity of several Gram-positive bacteria [78]. Inter-estingly, F. psychrophilum lacks the type III and IV secretionsystems usually present in Gram-negative pathogens; but,it has genes encoding PorT and PorR proteins, which areinvolved in transport and anchoring of virulence factorsof the bacteria [79, 80]. In addition, the F. psychrophilumgenome contains a large repertoire of genes involved inaerobic respiration, psychrotolerance, and stress response.

The sequencing of F. branchiophilum genome hasrevealed the existence of virulence mechanisms distinctlydifferent from the closest species, F. psychrophilum. The F.branchiophilum genome has the first cholera-like toxin in anonproteobacteria and an array of adhesins. A comparativeanalysis of its genome with genomes of other Flavobacteriumspecies revealed a smaller genome size, large differencesin chromosome organization, and fewer rRNA and tRNAgenes, fitting with its more fastidious growth. In addition,identification of certain virulence factors, genomic islands,and CRISPR (clustered regularly interspaced short palin-dromic repeats) systems points to the adaptive evolution ofF. branchiophilum by horizontal acquisition of genes.

6. Edwardsiella

The genus Edwardsiella belongs to subgroup 3 of γ-proteobacteria, encompassing a group of Gram-negativeenteric bacteria pathogenic to a variety of animals [81].Two very closely related species, Edwardsiella tarda andE. ictaluri are important fish pathogens. Both are Gram-negative motile rods that are cytochrome oxidase negativeand ferment glucose with production of acid and gas. Thetwo species can be differentiated biochemically in that E.tarda produces both indol and hydrogen sulfide, whereasE. ictaluri produces neither. Moreover, the two species donot cross-react serologically. E. tarda has been isolated frommany warm water fishes and some coldwater fishes, whereasE. ictaluri has been isolated only from a few species ofwarm water fishes (Table 2). Additionally, E. tarda causesdisease in such other animals as marine mammals, pigs,turtles, alligators, ostriches, skunks, and snakes [81]. It hasalso occasionally infected humans [82, 83]. In contrast,E. ictaluri is limited to fish, and survivors of epizooticsprobably become carriers. The geographic range of E. tardais worldwide, whereas that of E. ictaluri is still confined to thecatfish growing areas in the United States [84].

E. tarda causes a disease condition in fish called systemichemorrhagic septicemia with swelling skin lesions as well asulcer and necrosis in internal organs such as liver, kidney,


spleen, and musculature [85]. It has the ability of invadingand multiplying in epithelial cells and macrophages in orderto subvert the host immune system and to survive in the fish[86].

E. ictaluri is the causative agent of enteric septicemiaof catfish (ESC), a major disease affecting the catfishindustry. The disease can manifest as an acute form that ischaracterized by hemorrhagic enteritis and septicemia and achronic disease that is characterized by meningoencephalitis[87]. Gross external symptoms include hemorrhages on thebody, especially around the mouth and fins. Other signsinclude pale gills, exophthalmia, and small ulcerations on thebody [84].

The whole genome sequencing of the two species hasrecently been completed and published allowing comparativegenomic analysis of these very important fish pathogens [88,89]. The genome sequencing of the two closely related speciesE. tarda and E. ictaluri has revealed a high level of genomicplasticity with a high content of mobile genetic elements, ISelements, genomic islands, phage-like products, integrases,or recombinases. E. ictaluri displays high biochemical homo-geneity with only one serotype, but possess many IS elementsin the genome. In addition, highly variable G+C contentand a large quantity of variable number of tandem repeats(VNTRs) or direct repeat sequences were identified in theE. tarda genome indicating the rapid genomic evolutionundergoing in the species [88]. An interesting feature is theidentification of insertion sequence IS Saen1 of Salmonellaenterica serovar Enteritidis [90] in both E. tarda EIB202and E. ictaluri 93–146 genomes. Conversely, the differencein genomic islands among the three species may partiallyexplain their rapid evolutionary changes and diverginglineage from a common ancestor.

The E. tarda genome has a gene cluster sharing highsimilarities to the pvsABCDE-psuA-pvuA operon, whichencodes the proteins for the synthesis and utilization ofvibrioferrin, an unusual type of siderophore requiring nonri-bosomal peptide synthetase (NRPS) independent synthetases(NIS) and usually mediating the iron uptake systems in V.parahaemolyticus and V. alginolyticus [91, 92]. But E. ictalurigenome lacks siderophore biosynthesis genes, even though itpossesses heme binding/transport genes.

E. tarda genome is smaller than that of E. ictaluri andother sequenced genomes of Enterobacteriaceae, justifyingthe hypothesis that E. tarda may not be present as a free livingmicroorganism in natural waters but multiply intracellularlyin protozoans and transmitted to fish, reptile, and otheranimals or humans [81].

The E. tarda and E. ictaluri genomes have a multitudeof virulence factors including P pilus, type 1 fimbriae,nonfimbrial adhesins, invasins and hemagglutinins and var-ious secretion pathways including sec-dependent transportsystem, the components of the main terminal branch of thegeneral secretory pathway (GSP), the signal recognition par-ticle (SRP), and the sec-independent twin arginine transport(Tat), T1SS, TTSS, and T6SS indicating their evolutionaryfitness and ability to adapt to a variety of demandingecological niches and harsh host intracellular environments.

7. Yersinia ruckeri

Yersinia ruckeri, the causal agent of enteric redmouth (ERM)disease, which is a systemic bacterial infection of fishes, butis principally known for its occurrence in rainbow trout,Salmo gairdneri [93]. Y. ruckeri was initially isolated fromrainbow trout in the Hagerman Valley, Idaho, USA, in the1950s [94] and is now widely found in fish populationsthroughout North America, Australia, South Africa, andEurope [95]. Outbreaks of ERM usually begin with lowmortalities which slowly escalate and may result in highlosses. The problem may become large-scaled if chronicallyinfected fish are exposed to stressful conditions such as highstocking densities and poor water quality [96]. Y. ruckeriis a nonspore-forming bacterium which does not possess acapsule, but often has a flagellum [97].

Historically, Y. ruckeri is fairly homogenous in biochem-ical reactions. However, Y. ruckeri strains have recently beengrouped into clonal types on the basis of biotype, serotype,and outer membrane protein (OMP) profiles [98]. Strainsof serovars I and II [99], equivalent to serotypes O1a andO2b, respectively [100], cause most epizootic outbreaks incultured salmonids, serovar I being predominant in rainbowtrout [101]. Within serovar I, six clonal OMP types havebeen recognized, but only two are associated with majordisease outbreaks: clonal group 5, which includes the so-called Hagerman strain and clonal group 2 [98, 102]. Clonalgroup 5 comprises the majority of isolates, all of them motileand with a widespread distribution (Europe, North America,and South Africa). Clonal group 2 includes only nonmotilestrains isolated in the UK.

More recently, multilocus sequence typing has revealeddistinct phylogenetic divergence of Y. ruckeri from the restof the Yersinia genus raising doubts about its taxonomicposition [103]. This view has gained credibility after thegenome sequencing of Y. ruckeri, which has a substantiallyreduced total genome size (3.58 to 3.89 Mb), compared withthe 4.6 to 4.8 Mb seen in the genus generally [104]. Inaddition, Y. ruckeri was found to be the most evolutionarilydistant member of the genus with a number of featuresdistinct from other members of the genus.

Several common Yersinia genes were missing in Y. ruckeri.These included genes involved in xylose utilization, ureaseactivity, B12-related metabolism, and the mtnKADCBEUgene cluster that comprises the majority of the methioninesalvage pathway [104]. The genomic reduction achieved bylosing these and other genes is suggestive of its means ofadaptation to an obligatory life style in fish hosts.

8. Renibacterium salmoninarum

Renibacterium salmoninarum is a small Gram-positivediplobacillus, and the causative agent of bacterial kidneydisease (BKD), which is a slowly progressive, systemicinfection in salmonid fishes with a protracted course andan insidious nature [105]. The pathogen can be transmittedfrom fish to fish [106] or from adults to their progeny via eggs[107]. Infected fish may take months to show signs of disease.bacterial kidney disease is one of the most difficult bacterial


diseases of fish to treat [108], mainly due to its ability toevade phagocytosis and invade and survive in host cells[109, 110]. R. salmoninarum is very slow growing, and it isextremely difficult to apply genetic manipulation techniquesto study its gene functions.

R. salmoninarum, despite being an obligate intracellularpathogen of fish, is phylogenetically closest to the non-pathogenic environmental Arthrobacter species [51]. Basedon 16S rRNA phylogenetic analysis, R. salmoninarum hasbeen included in the actinomycetes subdivision and wasfound related to a subgroup harboring morphologicallyand chemotaxonomically rather heterogeneous taxa, includ-ing Arthrobacter, Micrococcus, Cellulomonas, Jonesia, Promi-cromonospora, Stomatococcus, and Brevibacterium [111]. Infact, Arthrobacter davidanieli is commercially used as avaccine (commercially known as Renogen) and can providesignificant cross-protection in Atlantic salmon, though notin Pacific salmon [112]. The genome sequencing of R.salmoninarum ATCC 33209 strain and two Arthrobacterstrains, the TC1 and FB24, has revealed many interestingaspects of how this obligates fish pathogen evolved, viagenomic reduction and horizontal gene acquisition, frommembers of the nonpathogenic genus Arthrobacter [51, 113].A total of 1562 ORF clusters were similar in R. salmoninarumand Arthrobacter spp. demonstrating the genetic basis for theefficiency and cross-protection of the A. davidanieli vaccine.

There is significant genome reduction in R. salmoni-narum genome, which is 1.44 Mb smaller than the chromo-some of TC1 and 1.55 Mb smaller than the chromosomeof FB24. The two Arthrobacter strains have several largeplasmids that are not present in the ATCC 33209 strain. Inaddition, these plasmids do not have high levels of similarityto sequences in the R. salmoninarum chromosome [51].

The presence of many IS elements, pseudogenes, andgenomic islands in R. salmoninarum genome coupled witha lack of restriction-modification systems contribute to theextensive disruption of ORFs as a strategy to reduce manypathways in the bacteria. Moreover, the highly homogeneousnature of R. salmoninarum with respect to the overallgenomic structure, biochemical properties, and surfaceantigens [114, 115] points to the evolution of this pathogentowards a strictly intracellular life style.

Several virulence factors including capsular synthesisgenes, heme acquisition operons, genes encoding possi-ble hemolysins, and the poorly characterized msa genesidentified in the R. salmoninarum genome seems to behorizontally acquired. Arthrobacter spp. lacks most of thesegene sequences, thus underlining the differential evolutionand adaptation of these two very closely related species tocontrasting ecological niches.

9. Streptococcus and Lactococcus

Gram-positive cocci belonging to the genera Streptococ-cus and Lactococcus are increasingly being recognized asimportant fish pathogens all over the world [116]. Thereare several different species of Gram-positive cocci, includ-ing Streptococcus parauberis, S. iniae, S. agalactiae (syn.

Streptococcus difficilis), S. phocae [117, 118], Lactococcusgarvieae (syn. Enterococcus seriolicida) [119], L. piscium[120–123], Vagococcus salmoninarum, and Carnobacteriumpiscicola [124], implicated in infectious diseases of warmwater as well as cold water fishes.

Streptococcosis appears to have very few limitationsin regard to geographic boundaries or host range, withoutbreaks occurring in aquaculture facilities worldwide andin many different cultured species. S. iniae, S.parauberis, S.agalactiae, and L. garvieae are known as the major pathogensof streptococcosis and lactococcosis in Oncorhynchus mykiss,Seriola quinqueradiata, Siganus canaliculatus, and Tilapiaspp. [125]. Recently, S. iniae and L. garvieae are alsorecognized as emerging zoonotic pathogens, causing diseasesin both fish and human beings [23, 126].

S. iniae is a β-haemolytic, Gram-positive coccus thatcauses generalized septicaemia and meningoencephalitis ina variety of warm water fishes [127], whereas S. parauberis isan α-hemolytic, Gram-positive coccus, mainly pathogenic incultured turbot (Scophthalmus maximus) and olive flounder,Paralichthys olivaceus. L. garvieae causes a hyperacute andhaemorrhagic septicemia in fishes particularly during thesummer time. General pathological symptoms of streptococ-cosis and lactococcosis in fishes are hemorrhage, congestion,lethargy, dark pigmentation, erratic swimming, and exoph-thalmos with clouding of the cornea [117, 128].

Complete genome sequences of different strains of S.parauberis and L. garvieae, important pathogenic speciesisolated from both fish and human, have been published[129–132].

S. parauberis is recognized as the dominant etiologicalagent of streptococcosis in fish [117], whereas both S.parauberis and S. uberis are involved the causation of bovinemastitis in dairy cow [133, 134].

S. parauberis is closer to S. uberis than with otherStreptococcus spp. and is biochemically and serologicallyindistinguishable from S. uberis [135]. Both species wereearlier considered as type I and II of S. uberis, but later shownto be phylogenetically distinct and renamed the type I as S.uberis and type II as S. parauberis [134].

The S. parauberis strain KCTC11537BP genome size fallsin the middle of the 1.8 to 2.3 Mb range of streptococcalgenomes sequenced to date and the average G+C content of35.6% is significantly lower than those of S. pyogenes [132].About 78% of genes are shared between the genomes of S.parauberis strain KCTC11537BP and S. uberis NC 012004,but they differ significantly at two regions of the genome,demonstrating the genomic basis for their separation intotwo species.

S. parauberis genome encodes an M-like protein ofS. iniae (SiM), which is an important virulence factorin S. iniae [136]. It also encodes hasA and hasB genesthat may be involved in capsule production for resistanceagainst phagocytosis. The genome analysis indicates that S.parauberis could possibly possess the ability to regulate themetabolism of more carbohydrates than other Streptococcusspecies and to synthesize all the aminoacids and regulatoryfactors required to adapt and survive in a highly hostile hostenvironment.


Complete genome sequences of L. garvieae strainUNIUD074, isolated from diseased rainbow trout in Italy, avirulent strain Lg2 (serotype KG2) and a nonvirulent strainATCC 49156 (serotype KG+), both isolated from diseasedyellowtail in Japan have recently been published [130, 131].In addition, genome sequence of L. garvieae strain 21881,isolated from a man suffering from septicemia has beenpublished [129].

The strains Lg2 and ATCC 49156 have 99% sequenceidentity and share 1944 orthologous genes, but are differentin 24 Lg2-specific genes that were absent in the ATCC49156 genome. One of the Lg2-specific genes is a 16.5 kbcapsule gene cluster, which confirms the earlier transmissionelectron microscopic finding that Lg2 is encapsulated, andATCC 49156 is nonencapsulated [137]. In fact, the capsulegene cluster has the features of a horizontally acquiredgenomic island conferring virulence to the Lg2 strain butmight have been lost from the ATCC 49156 strain whilesubculturing in the laboratory [131]. Both genomes carriedthree types of IS elements, prophage sequences, and integrasegenes and were found smaller than those of at least fivesequenced L. lactis genomes. The Lg2 genome lacks severalaminoacid biosynthesis genes, which is a characteristicfeature of pathogenic bacteria with reduced genomes. TheLg2 strain contains hemolysins, NADH oxidase and super-oxide dismutase (SOD), adhesins and sortase, which areknown virulence factors [137–139]. It also encodes a genefor phosphoglucomutase, a virulence factor conferring theresistance to peptide antimicrobials in S. iniae [140].

Although L. garvieae and L. lactis genomes share 75%CDS, about 25% genes are Lg2-specific hypothetical proteinsand proteins of unknown functions, which may be involvedin the virulence of the Lg2 strain. These findings indicatethat L. garvieae and L. lactis have significantly diverged fromthe common ancestor, and the L. garvieae is evolving into apathogenic species equipped with virulence features suitablefor living in the host environment.

10. Mycobacteria

Chronic infections in fish caused by different species ofmycobacteria have been well recognized [23, 141, 142].Several slow growing as well as fast growing species ofmycobacteria such as Mycobacterium marinum, M. fortui-tum, M. chelonae, and M. avium have been isolated fromwild and cultured fish suffering from mycobacteriosis indifferent parts of the world [143–145]. Among them, M.marinum is the most important fish pathogen, frequentlyisolated from a variety of fish species with granulomas[146]. It is also a known zoonotic pathogen, transmitted toman though fish handling in aquariums and aquaculturetanks, producing superficial and self-limiting lesions called“fish tank or aquarium tank granuloma” involving thecooler parts of the body such as hands, forearms, elbows,and knees [147, 148]. Although strain variation has beenreported [149], there is significant intraspecies sequencehomogeneity among different M. mrinum strains [150].However, it is hypothesized that only certain strains of M.marinum have zoonotic potential [151]. Phylogenetic studies

have shown that M. marinum is most closely related toM. ulcerans followed by M. tuberculosis [150]. Owing tothis, M. marinum and M. tuberculosis share many virulencefactors and significant pathological features and respond tosimilar antibiotics [152, 153]. Hence, M. marinum is alsoan important model organism to study the pathogenesis oftuberculosis [152, 153].

Interestingly, the genome of M. marinum is 50% biggerthan that of M. tuberculosis and seems to have acquired anumber of genes encoding NRPSs and the huge repertoireof PE, PPE, and ESX systems probably by HGT [154]. Bothspecies might have evolved differently from a common envi-ronmental mycobacteria. M. tuberculosis might have adaptedto its host intracellular life by extensive genome reductionand M. marinum, by and large retained or obtained genesrequired for its dual lifestyle and broad-host range.

11. Genome Sequencing to Find Novel Vaccineand Drug Targets in Fish Pathogens

Our understanding of the molecular basis of virulence ofcertain well-studied fish bacterial pathogens has increaseddramatically during the past decade. This has resulted fromthe application of recombinant DNA technology and cellbiology to investigate bacterial infections, and the develop-ment of genetic techniques for identifying virulence genes.

More recently, genome sequence information of severalbacterial fish pathogens has become available from genomesequencing projects. There is strong reason to believethat this understanding will be exploited to develop newinterventions against fish bacterial infections.

The relevance of sequencing projects for drug andvaccine discovery is obvious. During the “pregenomic” era,the vaccine candidate genes were individually identified bytedious gene knockout studies and virulence attenuation. Butnow, the complete genome sequencing provides informationon every virulence gene and all potential vaccine candidates,and the sequence databases will become indispensable forresearch in fish vaccinology and drug development.

After sequencing, the open reading frames (ORFs) aresearched against available databases for sequence similaritywith genes of known functions in other organisms. There areseveral strategies for gene annotation employing the tools ofpredictive bioinformatics programs combined with analysesof the published literature.

Multiple target vaccine candidate genes can be chosenand deleted simultaneously by various strategies includingglobal transposon mutagenesis and gene replacement tech-niques [155, 156] to study their effect on virulence andessentiality. A number of important virulence determinantsidentified in the sequenced genome can be targeted. Forexample, the sortase enzyme in Gram-positive fish pathogenswould be a very attractive universal vaccine and therapeuticdrug target, as it mediates covalent anchoring of many sur-face displayed antigenic and/or virulence related proteins inGram-positive bacteria [139]. The inactivation or inhibitionof the sortase enzyme can simultaneously prevent the surfacedisplay of a number of virulence factors, thus effectivelyattenuating the virulence of the pathogen [110, 157].


The availability of sequences of the complete surface anti-genic repertoire of pathogens, including protein and nopro-tein antigens would facilitate strategies for rational designof vaccines and drugs. In addition, the recent availabilityof large collections of the “virulogenome” of fish bacterialpathogens will provide enormous virulence sequence infor-mation for DNA vaccination studies. The whole complementof IS elements, prophages, and pathogenicity islands that canharbor virulence, and antimicrobial resistance gene clusterscan be easily identified in the genomes. The comparisonof genomes of different strains of the same bacteria orclosely related species can reveal how these strains or speciesbehave differently while infecting fish hosts, thus openingexciting opportunities for functional genomic analysis ofinfection processes and pathogenesis. However, experimentalvalidation of predicted functions of genes identified fromsequencing projects has lagged far behind the speed ofannotation, and the major challenge of researchers in thefield today is to understand the functional framework of thesequenced genomes.

12. Conclusions

There has been a steady increase in the number of speciesof bacteria implicated in fish diseases. The common fishpathogenic bacterial species belong to the genera Vibrio,Aeromonas, Flavobacterium, Yersinia, Edwardsiella, Strepto-coccus, lactococcus, Renibacterium, and Mycobacterium [23].However, there is growing indications that the pathogenicspecies spectrum as well as the geographic and host rangeis widening among fish pathogens [158–161], leading to theemergence of new pathogens. Unlike the situation in humanand animal medicine, fish diseases pose unique and dauntingchallenges. Fish are always bathed in a continuous medium ofwater, and fish disease treatment is essentially a populationmedicine. In addition, the current treatment methods arelargely ineffective, and the biology and genetics of mostfish bacterial pathogens are poorly understood, limiting theapplication of modern science-based pathogen interventionstrategies.

Rapid growth and expansion of genome sequencing ofhuman and animal pathogens enabled better understandingof their biology, evolution, and host adaptation strategies,and helped in combating many major diseases. Unfortu-nately, such developments and progress in the genomicsand functional genomics of fish pathogenic bacteria havebeen very slow. However, recent availability of cost-effectivehigh-throughput sequencing technologies has set the paceof sequencing of more fish pathogenic bacteria. Genomesequencing of a number of important bacterial pathogensof fish has helped us to better understand their biology andgenetics. The sequencing projects have unearthed excitingnew information on the adaptive evolution of fish pathogens,for example, how the nonpathogenic and ubiquitous soilbacteria such as Arthrobacter sp. has evolved into a strictlyobligate fish pathogen, R. salmoninarum, by shedding func-tional genes through genomic reduction to lead to a very cosyintracellular life style.

On the other hand, phenotypically similar strains ofthe same species differ in certain set of virulence geneclusters, acquired through HGT and become highly virulent.The capsule gene cluster in the L. garvieae Lg2 strainconfers virulence compared to noncapsulated ATCC 49156,which lacks the gene cluster. Nonpathogenic strains acquiregenomic islands from distantly related pathogenic speciesand emerge as new pathogens of fish.

Comparative pathogenomics of closely related bacteriahas increased our knowledge of how they vary in their viru-lence and their ability to adapt to different ecological niches.This is clearly evident in the difference in virulence of variousstrains of V. anguillarum and V. vulnificus, and among theclosely related species of the genus Flavobacterium. As morestrain-specific sequence information on bacterial pathogensof fish becomes available, we will have a better understandingof the subtle genomic differences among strains with varyingvirulence characteristics.

The typical pathogen evolutionary strategy of acquiring,shuffling and shedding genes mediated by IS elements,pseudogenes, prophage sequences, and HGT is also observedin most bacterial pathogens of fish. It is certain that thenew genomic information will bring paradigm changes inbacterial pathogenesis and should provide new perspectivesto our current thinking on the evolutionary and adaptivestrategies of aquatic bacteria and how they colonize andestablish in wider ecological niches and new host species.Moreover, the identification of key virulence factors inpathogenic strains should help us design efficient drugs andvaccines to combat major bacterial pathogens of fish.

However, it should be stressed that the genomic infor-mation will provide only a snapshot of the microorganism.Highly virulent clones armed with one or more acquiredvirulence factors can suddenly develop from the existingharmless microorganisms in the face of environmental,antibiotic, and host-induced selective pressures.

More intriguingly, about 40% of the genes in sequencedbacterial genomes constitute new putative genes and hypo-thetical proteins with mysterious functions and are con-served among several different species of bacteria. Evenin Escherichia coli, the most studied of all bacteria, only54% genes have currently been functionally characterizedbased on experimental evidence [162]. A close scrutiny ofthe sequenced genomes of fish pathogens reveals that theabove situation is essentially true for these pathogens aswell. Although current advances in functional genomics,structural genomics and bioinformatics have contributedimmensely to deciphering and extracting useful biologicalinformation from the vast genomic data, understandingand assigning functionality to the unique and new genesequences discovered in the genomes will be the major taskof genome biologists in the coming years.

Acknowledgments

This work was financially supported by various researchfundings from the Ministry of Agriculture and FisheriesWealth, Oman.


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